Response Gene to Complement 32 Maintains Blood Pressure Homeostasis by Regulating α-Adrenergic Receptor Expression.

RATIONALE: Hypertension prevalence is much higher among children and adolescents with low birth weight and greater postnatal weight gain than in individuals with normal birth weight. However, the cause and molecular mechanisms underlying this complication remain largely unknown. Our previous studies have shown that RGC-32 (response gene to complement 32)-deficient (RGC-32-/-) mice are born significantly smaller but grow faster than their WT (wild type) controls, which allows adult RGC-32-/- mice to attain body weights similar to those of control mice. OBJECTIVE: The objective of this study is to determine whether RGC-32-/- mice develop hypertension, and if so, to elucidate the underlying mechanisms. METHODS AND RESULTS: By using a radiotelemetry system, we found that RGC-32-/- mice exhibit higher mean arterial pressure than WT mice (101±4 versus 119±5 mm Hg), which enabled us to use RGC-32-/- mice to study the mechanisms underlying low birth weight-related hypertension. The increased blood pressure in RGC-32-/- mice was associated with increased vascular tone and decreased distensibility of small resistance arteries. The increased vascular tone was because of an increase in the relative contribution of sympathetic versus parasympathetic activity and was linked to increased expression of AT1R (angiotensin II type I receptor) and α1-AdR (α1-adrenergic receptor) in arterial smooth muscles. Mechanistically, RGC-32 regulated AT1R gene transcription by interacting with Sp1 (specificity protein 1) transcription factor and further blocking its binding to the AT1R promoter, leading to suppression of AT1R expression. The attenuation of AT1R leads to reduction in α1-AdR expression, which was critical for the balance of sympathetic versus parasympathetic control of vascular tone. Of importance, downregulation of RGC-32 in arterial smooth muscles was also associated with low birth weight and hypertension in humans. CONCLUSIONS: Our results indicate that RGC-32 is a novel protein factor vital for maintaining blood pressure homeostasis, especially in individuals with low birth weight.

Juvenile growth reduces the influence of epithelial sodium channels on myogenic tone in skeletal muscle arterioles.

Previous studies have documented that rapid juvenile growth is accompanied by functional changes in the arteriolar endothelium, but much less is known about functional changes in arteriolar smooth muscle over this period. In this study, we investigate the possible contribution of epithelial sodium channels (ENaC) to the myogenic behaviour of arterioles at two stages of juvenile growth. The effects of the ENaC inhibitor benzamil on different levels of myogenic tone were studied in isolated gracilis muscle arterioles from rats aged 21-28 days ("weanlings") and 42-49 days ("juveniles"). ENaC subunit expression in the arteriolar wall was also determined, and the interaction between ENaC and nitric oxide (NO) in regulating vascular tone was explored by combined use of benzamil and NG -monomethyl-l-arginine (l-NMMA). At physiological pressures, both steady-state myogenic tone and the dynamic adjustments in this tone triggered by acute pressure changes were less in juvenile arterioles than in weanling arterioles. α, ß and γ ENaC protein was present in arterioles at both ages, but benzamil only had an effect on myogenic tone in weanling arterioles. In these vessels, benzamil increased, rather than decreased, myogenic tone, and this effect was prevented by l-NMMA or endothelial removal. These findings suggest that although ENaC is present in gracilis muscle arterioles of both weanling and juvenile rats, it is not obligatory for the genesis of myogenic activity in these vessels at either age. However, ENaC activity can significantly modulate the level of myogenic tone through stimulation of endothelial NO release at an early stage of growth.

Salt, Angiotensin II, Superoxide, and Endothelial Function.

Proper function of the vascular endothelium is essential for cardiovascular health, in large part due to its antiproliferative, antihypertrophic, and anti-inflammatory properties. Crucial to the protective role of the endothelium is the production and liberation of nitric oxide (NO), which not only acts as a potent vasodilator, but also reduces levels of reactive oxygen species, including superoxide anion (O2•-). Superoxide anion is highly injurious to the vasculature because it not only scavenges NO molecules, but has other damaging effects, including direct oxidative disruption of normal signaling mechanisms in the endothelium and vascular smooth muscle cells. The renin-angiotensin system plays a crucial role in the maintenance of normal blood pressure. This function is mediated via the peptide hormone angiotensin II (ANG II), which maintains normal blood volume by regulating Na+ excretion. However, elevation of ANG II above normal levels increases O2•- production, promotes oxidative stress and endothelial dysfunction, and plays a major role in multiple disease conditions. Elevated dietary salt intake also leads to oxidant stress and endothelial dysfunction, but these occur in the face of salt-induced ANG II suppression and reduced levels of circulating ANG II. While the effects of abnormally high levels of ANG II have been extensively studied, far less is known regarding the mechanisms of oxidant stress and endothelial dysfunction occurring in response to chronic exposure to abnormally low levels of ANG II. The current article focuses on the mechanisms and consequences of this less well understood relationship among salt, superoxide, and endothelial function.

The effect of high salt intake on endothelial function: reduced vascular nitric oxide in the absence of hypertension.

Within the last 25 years, it has become increasingly clear that high dietary salt intake represents a risk factor for the development of cardiovascular disease that is independent of its well-known ability to increase arterial pressure in some individuals. Studies in normotensive experimental animals and human subjects have revealed that a key feature of this pressure-independent effect of dietary salt is a profound reduction in vascular nitric oxide (NO) bioavailability that limits endothelium-dependent dilation. This reduction in NO is strongly associated with increased levels of reactive oxygen species (ROS) generated by NAD(P)H oxidase, xanthine oxidase or uncoupled endothelial NO synthase within the vascular wall, leading not only to scavenging of NO but also to disruption of some signaling pathways that mediate its production. The mechanistic link between high salt intake and elevated levels of enzymatically generated ROS in the peripheral vasculature is not clear, but a reduction in circulating angiotensin II may play a key role in this regard. Additional studies are needed to further elucidate the mechanisms, both at the systemic level and within the vascular wall, that trigger these salt-induced deficits in endothelial function, and to further clarify how the attendant loss of NO may disrupt tissue blood flow regulation and ultimately lead to adverse cardiovascular events.

Changes in eNOS phosphorylation contribute to increased arteriolar NO release during juvenile growth.

Nitric oxide (NO) mediates a major portion of arteriolar endothelium-dependent dilation in adults, but indirect evidence has suggested that NO contributes minimally to these responses in the young. Isolated segments of arterioles were studied in vitro to verify this age-related increase in NO release and investigate the mechanism by which it occurs. Directly measured NO release induced by ACh or the Ca(2+) ionophore A-23187 was five- to sixfold higher in gracilis muscle arterioles from 42- to 46-day-old (juvenile) rats than in those from 25- to 28-day-old (weanling) rats. There were no differences between groups in arteriolar endothelial NO synthase (eNOS) expression or tetrahydrobiopterin levels, and arteriolar l-arginine levels were lower in juvenile vessels than in weanling vessels (104 ± 6 vs.126 ± 3 pmol/mg). In contrast, agonist-induced eNOS Thr(495) dephosphorylation and eNOS Ser(1177) phosphorylation (events required for maximal activity) were up to 30% and 65% greater, respectively, in juvenile vessels. Juvenile vessels did not show increased expression of enzymes that mediate these events [protein phosphatases 1 and 2A and PKA and PKB (Akt)] or heat shock protein 90, which facilitates Ser(1177) phosphorylation. However, agonist-induced colocalization of heat shock protein 90 with eNOS was 34-66% greater in juvenile vessels than in weanling vessels, and abolition of this difference with geldanamycin also abolished the difference in Ser(1177) phosphorylation between groups. These findings suggest that growth-related increases in arteriolar NO bioavailability may be due at least partially to changes in the regulation of eNOS phosphorylation and increased signaling activity, with no change in the abundance of eNOS signaling proteins.

Pulmonary particulate matter and systemic microvascular dysfunction.

Pulmonary particulate matter (PM) exposure has been epidemiologically associated with an increased risk of cardiovascular morbidity and mortality, but the mechanistic foundations for this association are unclear. Exposure to certain types of PM causes changes in the vascular reactivity of several macrovascular segments. However, no studies have focused upon the systemic microcirculation, which is the primary site for the development of peripheral resistance and, typically, the site of origin for numerous pathologies. Ultrafine PM--also referred to as nanoparticles, which are defined as ambient and engineered particles with at least one physical dimension less than 100 nm (Oberdorster et al. 2005)--has been suggested to be more toxic than its larger counterparts by virtue of a larger surface area per unit mass. The purpose of this study was fourfold: (1) determine whether particle size affects the severity of postexposure microvascular dysfunction; (2) characterize alterations in microvascular nitric oxide (NO) production after PM exposure; (3) determine whether alterations in microvascular oxidative stress are associated with NO production, arteriolar dysfunction, or both; and (4) determine whether circulating inflammatory mediators, leukocytes, neurologic mechanisms, or a combination of these play a fundamental role in mediating pulmonary PM exposure and peripheral microvascular dysfunction. To achieve these goals, we created an inhalation chamber that generates stable titanium dioxide (TiO2) aerosols at concentrations up to 20 mg/m3. TiO2 is a well-characterized particle devoid of soluble metals. Sprague Dawley and Fischer 344 (F-344) rats were exposed to fine or nano-TiO2 PM (primary count modes of approximately 710 nm and approximately 100 nm in diameter, respectively) at concentrations of 1.5 to 16 mg/m3 for 4 to 12 hours to produce pulmonary loads of 7 to 150 microg in each rat. Twenty-four hours after pulmonary exposure, the following procedures were performed: the spinotrapezius muscle was prepared for in vivo microscopy, blood samples were taken from an arterial line, and various tissues were harvested for histologic and immunohistochemical analyses. Some rats received a bolus dose of cyclophosphamide 3 days prior to PM exposure to deplete circulating neutrophils and bronchoalveolar lavage (BAL) was performed in separate groups of rats exposed to identical TiO2 loads. No significant differences in BAL fluid composition based on PM size or load were found in these rats. Plasma levels of interleukin (IL)-2, IL-18, IL-13, and growth-related oncogene (GRO) (also known as keratinocyte-derived-chemokine [KC]) were altered after PM exposure. In rats exposed to fine TiO2, endothelium-dependent arteriolar dilation was significantly decreased, and this dysfunction was robustly augmented in rats exposed to nano-TiO2. This effect was not related to an altered smooth-muscle responsiveness to NO because arterioles in both groups dilated comparably in response to the NO donor sodium nitroprusside (SNP). Endogenous microvascular NO production was similarly decreased after inhalation of either fine or nano-TiO2 in a dose-dependent manner. Microvascular oxidative stress was significantly increased among both exposure groups. Furthermore, treatment with antioxidants (2,2,6,6-tetramethylpiperdine-N-oxyl [TEMPOL] plus catalase), the myeloperoxidase (MPO) inhibitor 4-aminobenzoic hydrazide (ABAH), or the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) inhibitor apocynin partially restored NO production and normalized arteriolar function in both groups. Neutrophil depletion restored dilation in PM-exposed rats by as much as 42%. Coincubation of the spinotrapezius muscle with the fast sodium (Na+) channel antagonist tetrodotoxin (TTX) restored arteriolar dilation by as much as 54%, suggesting that sympathetic neural input may be affected by PM exposure. The results of these experiments indicate that (1) the size of inhaled PM dictates the intensity of systemic microvascular dysfunction; (2) this arteriolar dysfunction is characterized by a decreased bioavailability of endogenous NO; (3) the loss of bioavailable NO after PM exposure is at least partially caused by elevations in local oxidative stress, MPO activity, NADPH oxidase activity, or a combination of these responses; and (4) circulating neutrophils and sympathetic neurogenic mechanisms also appear to be involved in the systemic microvascular dysfunction that follows PM exposure. Taken together, these mechanistic studies support prominent hypotheses that suggest peripheral vascular effects associated with PM exposure are due to the activation of inflammatory mechanisms, neurogenic mechanisms, or both.

Salt modulates vascular response through adenosine A(2A) receptor in eNOS-null mice: role of CYP450 epoxygenase and soluble epoxide hydrolase.

High salt (HS) intake can change the arterial tone in mice, and the nitric oxide (NO) acts as a mediator to some of the receptors mediated vascular response. The main aim of this study was to explore the mechanism behind adenosine-induced vascular response in HS-fed eNOS(+/+) and eNOS(-/-) mice The modulation of vascular response by HS was examined using aortas from mice (eNOS(+/+) and eNOS(-/-)) fed 4% (HS) or 0.45% (NS) NaCl-diet through acetylcholine (ACh), NECA (adenosine-analog), CGS 21680 (A(2A) AR-agonist), MS-PPOH (CYP epoxygenase-blocker; 10(-5) M), AUDA (sEH-blocker; 10(-5) M), and DDMS (CYP4A-blocker; 10(-5) M). ACh-response was greater in HS-eNOS(+/+) (+59.3 ± 6.3%) versus NS-eNOS(+/+) (+33.3 ± 8.0%; P < 0.05). However, there was no response in both HS-eNOS(-/-) and NS-eNOS(-/-). NECA-response was greater in HS-eNOS(-/-) (+37.4 ± 3.2%) versus NS-eNOS(-/-) (+7.4.0 ± 3.8%; P < 0.05). CGS 21680-response was also greater in HS-eNOS(-/-) (+45.4 ± 5.2%) versus NS-eNOS(-/-)(+5.1 ± 5.0%; P < 0.05). In HS-eNOS(-/-), the CGS 21680-response was reduced by MS-PPOH (+7.3 ± 3.2%; P < 0.05). In NS-eNOS(-/-), the CGS 21680-response was increased by AUDA (+38.2 ± 3.3%; P < 0.05) and DDMS (+30.1 ± 4.1%; P < 0.05). Compared to NS, HS increased CYP2J2 in eNOS(+/+) (35%; P < 0.05) and eNOS(-/-) (61%; P < 0.05), but decreased sEH in eNOS(+/+) (74%; P < 0.05) and eNOS(-/-) (40%; P < 0.05). Similarly, CYP4A decreased in HS-eNOS(+/+) (35%; P < 0.05) and HS-eNOS(-/-) (34%; P < 0.05). These data suggest that NS causes reduced-vasodilation in both eNOS(+/+) and eNOS(-/-) via sEH and CYP4A. However, HS triggers possible A(2A)AR-induced relaxation through CYP epoxygenase in both eNOS(+/+) and eNOS(-/-).

Endothelium-dependent control of vascular tone during early postnatal and juvenile growth.

Endothelial dysfunction can develop at an early age in children with risk factors for cardiovascular disease. A clear understanding of the nature of this dysfunction and how it can worsen over time requires detailed information on the normal growth-related changes in endothelial function on which the pathological changes are superimposed. This review summarizes our current understanding of these normal changes, as derived from studies in four different mammalian species. Although the endothelium plays an important role in controlling vascular tone from birth onward, the vasoactive molecules that mediate this control often change during postnatal or juvenile growth. The specifics of this transition to an adult endothelial cell phenotype can vary depending on the vascular bed. During growth, the contribution of nitric oxide to endothelium-dependent dilation generally increases in the lung, cerebral cortex, and skeletal muscle, but decreases in the intestine. Endothelial capacity for release of other vasoactive factors (e.g., cyclooxygenase products, hydrogen peroxide, carbon monoxide) can also increase or decrease during growth. Although these changes have been well documented, there is less information on their underlying cellular or molecular events. Further research is required to clarify these mechanisms, and to evaluate the functional significance of such shifts in endothelial phenotype.

Modulation by salt intake of the vascular response mediated through adenosine A(2A) receptor: role of CYP epoxygenase and soluble epoxide hydrolase.

High-salt intake can change the effect of adenosine on arterial tone in mice. The aim of this study was to clarify the mechanism by which this occurs. Using aortas from mice fed a 4% NaCl (HS) or 0.45% NaCl (NS) diet for 4-5 wks, concentration-response curves for ACh, 5'-N-ethylcarboxamidoadenosine (NECA; adenosine analog) and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride hydrate [CGS-21680; A(2A) adenosine receptor (A(2A) AR) agonist] were obtained with N(omega)-nitro-L-arginine methyl ester (L-NAME; nitric oxide inhibitor, 10(-4) M), methylsulfonyl-propargyloxyphenylhexanamide [MS-PPOH; a CYP (cytochrome P-450) epoxygenase blocker, 10(-5) M including CYP2J2], 12-(3-adamantan-1-yl-ureido)dodecanoic acid [AUDA; soluble epoxide hydrolase (sEH) blocker, 10(-5) M], dibromo-dodecenyl-methylsulfimide [DDMS; CYP omega-hydroxylase (CYP4A blocker), 10(-5) M], glibenclamide (K(ATP) channel blocker; 10(-5) M) and 5-hydroxydecanoate (5-HD; mitochondrial-K(ATP) channel blocker, 10(-4) M). HS dose response to ACh (10(-7) - 10(-5) M) was not different from NS (P > 0.05). Relaxation to 10(-6) M NECA was greater in the HS group (28.4 +/- 3.9%) than in the NS group (4.1 +/- 2.3%). Relaxation to 10(-6) M CGS-21680 was also greater in HS (27.9 +/- 4.5%) than in NS (4.9 +/- 2.2%). L-NAME was able to block the dose response of ACh (10(-7) - 10(-5) M) equally in both HS and NS (P > 0.05), whereas L-NAME did not block CGS-21680-induced response in HS. In HS the CGS-21680 response was greatly reduced by MS-PPOH (to 4.7 +/- 2.0%) and 5-HD (to 8.9 +/- 2.2%), and also abolished by glibenclamide (-1.0 +/- 5.9%). In NS, the CGS-21680 response was increased by AUDA (to 26.3 +/- 3.4%) and DDMS (to 27.2 +/- 3.0%). Compared with NS, HS vessels showed increased CYP2J2 and A(2A) AR expression (46 and 74% higher, respectively) but decreased sEH, CYP4A, and A(1) AR expression (75, 30, and 55% lower, respectively). These data suggest that in mice fed NS-containing diet, upregulation of arterial A(1) receptor causes vasoconstriction via increased sEH and CYP4A proteins. However, in mice fed HS-containing diet, upregulation of A(2A) receptor protein triggers vascular relaxation through ATP-sensitive (K(+)) channels via upregulation of CYP2J2 enzyme.

Decreased arteriolar tetrahydrobiopterin is linked to superoxide generation from nitric oxide synthase in mice fed high salt.

OBJECTIVE: Impaired endothelium-dependent arteriolar dilation in mice fed high salt (HS) is due to local oxidation of nitric oxide (NO) by superoxide anion (O(2) (-)). We explored the possibility that "uncoupled" endothelial nitric oxide synthase (eNOS) is the source of this O(2) (-). METHODS: Levels of L-arginine (L-Arg), tetrahydrobiopterin (BH(4)), and O(2) (-) (hydroethidine oxidation) were measured in spinotrapezius muscle arterioles of mice fed normal salt (0.45%, NS) or (4%, HS) diets for 4 weeks, with or without dietary L-Arg supplementation. The contribution of NO to endothelium-dependent dilation was determined from the effect of N(omega)-nitro-L-arginine methyl ester (L-NAME) on responses to acetylcholine (ACh). RESULTS: Arterioles in HS mice had lower [BH(4)] and higher O(2) (-) levels than those in NS mice. ACh further increased arteriolar O(2) (-) in HS mice only. L-Arg supplementation prevented the reduction in [BH(4)] in arterioles of HS mice, and O(2) (-) was not elevated in these vessels. Compared to NS mice, arteriolar ACh responses were diminished and insensitive to L-NAME in HS mice, but not in HS mice supplemented with L-Arg. CONCLUSIONS: These findings suggest that eNOS uncoupling due to low [BH(4)] is responsible for O(2) (-) generation and reduced NO-dependent dilation in arterioles of mice fed a HS diet.

Growth-dependent changes in the contribution of carbon monoxide to arteriolar function.

BACKGROUND/AIMS: Endothelium-dependent dilation of skeletal muscle arterioles is mediated by unknown factors in very young rats. We assessed the possible contribution of carbon monoxide (CO) to this dilation and to dilation in older animals. METHODS: The effects of de-endothelialization or various pharmacological inhibitors on responses to CO or endothelium-dependent dilators were studied in gracilis muscle arterioles from rats at 3-4 weeks ('weanlings') and 6-7 weeks ('juveniles'). RESULTS: Exogenous CO constricted, rather than dilated, arterioles from both age groups. This constriction was reduced by endothelial removal or NOS inhibition in juvenile, but not weanling, arterioles. In contrast, this constriction was abolished by K(+) channel inhibition in weanling, but not juvenile, arterioles. The heme precursor delta-aminolevulinic acid constricted juvenile arterioles but did not affect weanling arterioles. The heme oxygenase inhibitor chromium (III) mesoporphyrin IX abolished the endothelium-dependent dilation of juvenile arterioles to simvastatin, and reduced ACh- and simvastatin-induced dilations of weanling arterioles. CONCLUSION: These findings suggest that relatively high concentrations of exogenous CO can cause constriction by inhibiting endothelium-derived NO in juvenile arterioles and inhibiting K(+) channels in weanling arterioles. Endogenous CO produced at lower concentrations can contribute to endothelium-dependent dilation in both age groups.

Integration of skeletal muscle resistance arteriolar reactivity for perfusion responses in the metabolic syndrome.

Previous study suggests that with evolution of the metabolic syndrome, patterns of arteriolar reactivity are profoundly altered and may constrain functional hyperemia. This study investigated interactions between parameters of vascular reactivity at two levels of resistance arterioles in obese Zucker rats (OZR), translating these observations into perfusion regulation for in situ skeletal muscle. Dilation of isolated and in situ resistance arterioles from OZR to acetylcholine, arachidonic acid (AA), and hypoxia (isolated arterioles only) were blunted vs. lean Zucker rats (LZR), although dilation to adenosine was intact. Increased adrenergic tone (phenylephrine) or intralumenal pressure (ILP) impaired dilation in both strains (OZR>LZR). Treatment of OZR arterioles with Tempol (superoxide dismutase mimetic) or SQ-29548 (prostaglandin H2/thromboxane A2 receptor antagonist) improved dilator reactivity under control conditions and with increased ILP, but had minimal effect with increased adrenergic tone. Arteriolar dilation to adenosine was well maintained in both strains under all conditions. For in situ cremasteric arterioles, muscle contraction-induced elevations in metabolic demand elicited arteriolar dilations and hyperemic responses that were blunted in OZR vs. LZR, although distal parallel arterioles were characterized by heterogeneous dilator and perfusion responses. alpha-Adrenoreceptor blockade improved outcomes at rest but had minimal effect with elevated metabolic demand. Treatment with Tempol or SQ-29548 had minimal impact at rest, but lessened distal arteriolar perfusion heterogeneity with increased metabolic demand. In blood-perfused gastrocnemius of OZR, perfusion was constrained primarily by adrenergic tone, while myogenic activation and endothelium-dependent dilation did not appear to contribute significantly to ischemia. These results of this novel, integrated approach suggest that adrenergic tone and metabolic dilation are robust determinants of bulk perfusion to skeletal muscle of OZR, while endothelial dysfunction may more strongly regulate perfusion distribution homogeneity via the impact of oxidant stress and AA metabolism.

Pulmonary nanoparticle exposure disrupts systemic microvascular nitric oxide signaling.

We have shown that pulmonary nanoparticle exposure impairs endothelium dependent dilation in systemic arterioles. However, the mechanism(s) through which this effect occurs is/are unclear. The purpose of this study was to identify alterations in the production of reactive species and endogenous nitric oxide (NO) after nanoparticle exposure, and determine the relative contribution of hemoproteins and oxidative enzymes in this process. Sprague-Dawley rats were exposed to fine TiO2 (primary particle diameter approximately 1 microm) and TiO2 nanoparticles (primary particle diameter approximately 21 nm) via aerosol inhalation at depositions of 4-90 microg per rat. As in previous intravital experiments in the spinotrapezius muscle, dose-dependent arteriolar dilations were produced by intraluminal infusions of the calcium ionophore A23187. Nanoparticle exposure robustly attenuated these endothelium-dependent responses. However, this attenuation was not due to altered microvascular smooth muscle NO sensitivity because nanoparticle exposure did not alter arteriolar dilations in response to local sodium nitroprusside iontophoresis. Nanoparticle exposure significantly increased microvascular oxidative stress by approximately 60%, and also elevated nitrosative stress fourfold. These reactive stresses coincided with a decreased NO production in a particle deposition dose-dependent manner. Radical scavenging, or inhibition of either myeloperoxidase or nicotinamide adenine dinucleotide phosphate oxidase (reduced) oxidase partially restored NO production as well as normal microvascular function. These results indicate that in conjunction with microvascular dysfunction, nanoparticle exposure also decreases NO bioavailability through at least two functionally distinct mechanisms that may mutually increase local reactive species.

High-salt diet enhances mouse aortic relaxation through adenosine A2A receptor via CYP epoxygenases.

We hypothesize that A(2A) adenosine receptors (A(2A) AR) promote aortic relaxation in mice through cytochrome P450 (CYP)-epoxygenases and help to avoid salt sensitivity. Aortas from male mice maintained on a high-salt (HS; 7% NaCl) or normal-salt (NS; 0.45% NaCl) diet for 4-5 wks were used. Concentration-response curves (10(-11)-10(-5) M) for 5'-N-ethylcarboxamidoadenosine (NECA; a nonselective adenosine analog) and CGS 21680 (A(2A) AR agonist) were obtained with different antagonists including ZM 241385 (A(2A) AR antagonist; 10(-6) M), SCH 58261 (A(2A) AR antagonist; 10(-6) M), N(omega)-nitro-l-arginine methyl ester (l-NAME; endothelial nitric oxide synthase inhibitor; 10(-4) M) and inhibitors including methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH; CYP epoxygenases inhibitor; 10(-5)M), 14,15-epoxyeicosa-5(z)-enoic acid (14,15-EEZE; EET antagonist; 10(-5)M), dibromo-dodecenyl-methylsulfimide (DDMS; CYP4A inhibitor; 10(-5)M), and HET0016 (20-HETE inhibitor; 10(-5)M). At 10(-7) M of NECA, significant relaxation in HS (+22.58 +/- 3.12%) was observed compared with contraction in NS (-10.62 +/- 6.27%, P < 0.05). ZM 241385 changed the NECA response to contraction (P < 0.05) in HS. At 10(-7) M of CGS 21680, significant relaxation in HS (+32.04 +/- 3.08%) was observed compared with NS (+10.45 +/- 1.34%, P < 0.05). SCH 58261, l-NAME, MS-PPOH, and 14,15-EEZE changed the CGS 21680-induced relaxation to contraction (P < 0.05) in HS. Interestingly, DDMS and HET0016 changed CGS 21680 response to relaxation (P < 0.05) in NS; however, there was no significant difference found between DDMS, HET0016-treated HS and NS vs. nontreated HS group (P > 0.05). CYP2C29 protein was 55% and 74% upregulated in HS vs. NS (P < 0.05) mice aorta and kidney, respectively. CYP4A protein was 30.30% and 35.70% upregulated in NS vs. HS (P < 0.05) mice aorta and kidneys, respectively. A(1) AR was downregulated, whereas A(2A) AR was upregulated in HS compared with NS. These data suggest that HS may activate CYP2C29 via A(2A) AR, causing relaxation, whereas NS may contribute to the upregulation of CYP4A causing contraction.

Increased myogenic responsiveness of skeletal muscle arterioles with juvenile growth.

Previous studies from this laboratory suggest that during juvenile growth, structural changes in the arteriolar network are accompanied by changes in some of the mechanisms responsible for regulation of tissue blood flow. To test the hypothesis that arteriolar myogenic behavior is altered with growth, we studied gracilis muscle arterioles isolated from Sprague-Dawley rats at two ages: 21-28 and 42-49 days. When studied at their respective in vivo pressures, the myogenic index (instantaneous slope of the active pressure-diameter curve) of arterioles from 42-49-day-old rats was more negative than that of arterioles from 21-28-day-old rats, indicating greater myogenic responsiveness. Endothelial denudation, or prostaglandin H(2) (PGH(2))/thromboxane A(2) (TxA(2)) receptor antagonism without denudation, significantly reduced the myogenic responsiveness of arterioles from the older rats over a wide range of pressures but had no consistent effects on the myogenic responsiveness of arterioles from the younger rats. The heme oxygenase inhibitor chromium (III) mesoporphyrin IX chloride had no effect on the myogenic activity of arterioles from either age group. These findings indicate that microvascular growth in young animals is accompanied by an increase in the myogenic behavior of arterioles, possibly because PGH(2) or TxA(2) assumes a role in reinforcing myogenic activity over this period. As a result, the relative contribution of myogenic activity to blood flow regulation in skeletal muscle may increase during rapid juvenile growth.

Hydrogen peroxide emerges as a regulator of tone in skeletal muscle arterioles during juvenile growth.

OBJECTIVE: The endothelium-dependent dilation of skeletal muscle arterioles is mediated by factors that have not been identified in young rats, and partly mediated by an unidentified hyperpolarizing factor in maturing rats. This study was designed to determine if endogenous hydrogen peroxide (H2O2) contributes to this arteriolar dilation at either of these growth stages. METHODS: Gracilis muscle arterioles were isolated from rats at ages 24-26 days ("weanlings") and 46-48 days ("juveniles"). We investigated the effects of catalase treatment on the endothelium-dependent dilation of these vessels to simvastatin and acetylcholine (ACh). Catalase-sensitive 2',7'-dichlorofluorescein (DCF) fluorescence also was measured as an index of H2O2 formation, and arteriolar dilation to exogenous H2O2 was pharmacologically probed in each age group. RESULTS: Responses to simvastatin and ACh were attenuated by catalase in juvenile, but not weanling, arterioles. Juvenile, but not weanling, arterioles also displayed catalase-sensitive DCF fluorescence that was increased by ACh. Exogenous H2O2 could induce dilation in juvenile, but not weanling, arterioles. In juvenile arterioles, this dilation was abolished by the K+ channel inhibitors TEA and glibenclamide, and attenuated by NOS inhibition or endothelial removal. CONCLUSIONS: These findings suggest that endogenous H2O2 contributes to endothelium-dependent arteriolar dilation in juvenile rats, but not in younger rats, and that H2O2 acts in juvenile rats by stimulating endothelial NO release and activating smooth muscle K+ channels.

Hydrogen peroxide-dependent arteriolar dilation in contracting muscle of rats fed normal and high salt diets.

OBJECTIVE: High dietary salt intake decreases the arteriolar dilation associated with skeletal muscle contraction. Because hydrogen peroxide (H2O2) can be released from contracting muscle fibers, this study was designed to assess the possible contribution of H2O2 to skeletal muscle functional hyperemia and its sensitivity to dietary salt. METHODS: The authors investigated the effect of catalase treatment on arteriolar dilation and hyperemia in contracting spinotrapezius muscle of rats fed a normal salt (0.45%, NS) or high salt (4%, HS) diet for 4 weeks. Catalase-sensitive 2',7'-dichlorofluorescein (DCF) fluorescence was measured as an index of H2O2 formation, and the mechanism of arteriolar dilation to H2O2 was probed in each group using pharmacological inhibitors. RESULTS: DCF fluorescence increased with muscle contraction, but not if catalase was present. Catalase also reduced arteriolar dilation and hyperemia during contraction in both dietary groups. Exogenous H2O2 dilated arterioles in both groups, with greater responses in HS rats. Guanylate cyclase inhibition did not affect arteriolar responses to H2O2 in either group, but K(Ca) or KATP channel inhibition equally reduced these responses, and K(ATP) channel inhibition equally reduced functional hyperemia in both groups. CONCLUSIONS: These results indicate that locally produced H2O2 contributes to arteriolar dilation and hyperemia in contracting skeletal muscle, and that the effect of H2O2 on arteriolar tone in this vascular bed is mediated largely through K+ channel activation. High dietary salt intake does not reduce the contribution of H2O2 to active hyperemia, or alter the mechanism through which H2O2 relaxes arteriolar smooth muscle.

Growth-dependent changes in endothelial factors regulating arteriolar tone.

Previous studies from this laboratory suggest that during maturation, rapid microvascular growth is accompanied by changes in the mechanisms responsible for regulation of tissue blood flow. To further define these changes, we studied isolated gracilis muscle arterioles from weanling ( approximately 25 days) and juvenile ( approximately 44 days) Sprague-Dawley rats to test the hypothesis that endothelial mechanisms for the control of arteriolar tone are altered with growth. Responses to the endothelium-dependent dilator acetylcholine (ACh) were greater in weanling arterioles (WA) than in juvenile arterioles (JA), whereas there were no consistent differences between age groups in arteriolar responses to other endothelium-dependent agonists (A-23187, vascular endothelial growth factor, and simvastatin). Inhibition of nitric oxide synthase (NOS) with N(omega)-nitro-l-arginine methyl ester (l-NAME) attenuated ACh-induced dilation in JA but not in WA. In JA, combined inhibition of NOS and cyclooxygenase (with indomethacin) reduced the dilator responses to ACh and simvastatin by approximately 90% and approximately 70%, respectively, but had no effect in WA. Cytochrome P450 epoxygenase inhibition [with 2-(propargyloxyphenyl) hexanoic acid] had no effect on responses to ACh or simvastatin in either age group. Inhibition of Ca(2+)-activated or ATP-dependent potassium channels (with tetraethylammonium or glibenclamide, respectively) reduced these arteriolar responses in JA but not those in WA. These findings suggest that in fully grown microvascular networks, endothelium-dependent arteriolar dilation is mediated by the combined release of endothelial nitric oxide and vasodilator prostanoids, and in part through activation of Ca(2+)-activated and ATP-dependent potassium channels. However, during earlier microvascular growth, this dilation is mediated by other factors yet to be identified. This may have significant implications for the regulation of tissue perfusion during microvascular development.

High dietary salt reduces the contribution of 20-HETE to arteriolar oxygen responsiveness in skeletal muscle.

The coupling of tissue blood flow to cellular metabolic demand involves oxygen-dependent adjustments in arteriolar tone, and arteriolar responses to oxygen can be mediated, in part, by changes in local production of 20-HETE. In this study, we examined the long-term effect of dietary salt on arteriolar oxygen responsiveness in the exteriorized, superfused rat spinotrapezius muscle and the role of 20-HETE in this responsiveness. Rats were fed either a normal-salt (NS, 0.45%) or high-salt (HS, 4%) diet for 4-5 wk. There was no difference in steady-state tissue Po(2) between NS and HS rats, and elevation of superfusate oxygen content from 0% to 10% caused tissue Po(2) to increase by the same amount in both groups. However, the resulting reductions in arteriolar diameter and blood flow were less in HS rats than NS rats. Inhibition of 20-HETE formation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or 17-octadecynoic acid (17-ODYA) attenuated oxygen-induced constriction in NS rats but not HS rats. Exogenous 20-HETE elicited arteriolar constriction that was greatly reduced by the large-conductance Ca(2+)-activated potassium (K(Ca)) channel inhibitors tetraethylammonium chloride (TEA) and iberiotoxin (IbTx) in NS rats and a smaller constriction that was less sensitive to TEA or IbTx in HS rats. Arteriolar responses to exogenous angiotensin II were similar in both groups but more sensitive to inhibition with DDMS in NS rats. Norepinephrine-induced arteriolar constriction was similar and insensitive to DDMS in both groups. We conclude that 20-HETE contributes to oxygen-induced constriction of skeletal muscle arterioles via inhibition of K(Ca) channels and that a high-salt diet impairs arteriolar responses to increased oxygen availability due to a reduction in vascular smooth muscle responsiveness to 20-HETE.

High salt intake reduces endothelium-dependent dilation of mouse arterioles via superoxide anion generated from nitric oxide synthase.

In skeletal muscle arterioles of normotensive rats fed a high salt diet, the bioavailability of endothelium-derived nitric oxide (NO) is reduced by superoxide anion. Because the impact of dietary salt on resistance vessels in other species is largely unknown, we investigated endothelium-dependent dilation and oxidant activity in spinotrapezius muscle arterioles of C57BL/6J mice fed normal (0.45%, NS) or high salt (7%, HS) diets for 4 wk. Mean arterial pressure in HS mice was not different from that in NS mice, but the magnitude of arteriolar dilation in response to different levels of ACh was 42-57% smaller in HS mice than in NS mice. Inhibition of nitric oxide synthase (NOS) with N(G) monomethyl L-arginine (L-NMMA) significantly reduced resting diameters and reduced responses to ACh (by 45-63%) in NS mice but not in HS mice. Arteriolar wall oxidant activity, as assessed by tetranitroblue tetrazolium reduction or hydroethidine oxidation, was greater in HS mice than in NS mice. Exposure to the superoxide scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) + catalase reduced this oxidant activity to normal and restored normal arteriolar responsiveness to ACh in HS mice but had no effect in NS mice. L-NMMA also restored arteriolar oxidant activity to normal in HS mice. ACh further increased arteriolar oxidant activity in HS mice but not in NS mice, and this effect was prevented with L-NMMA. These data suggest that a high salt diet promotes increased generation of superoxide anion from NOS in the murine skeletal muscle microcirculation, thus impairing endothelium-dependent dilation through reduced NO bioavailability.